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1 Genetic divergence and evolutionary relationships in six species of genera 2 Hoplobatrachus and Euphlyctis (Amphibia: Anura) from Bangladesh and other 3 Asian countries revealed by mitochondrial gene sequences 4 5 Mohammad Shafiqul Alam a, Takeshi Igawa a, Md. Mukhlesur Rahman Khan b, 6 Mohammed Mafizul Islam a, Mitsuru Kuramoto c, Masafumi Matsui d, Atsushi 7 Kurabayashi a, Masayuki Sumida a* 8 9 a Institute for Amphibian Biology, Graduate School of Science, Hiroshima 10 University, Higashihiroshima 739-8526, Japan 11 b Bangladesh Agricultural University, Mymensingh-2202, Bangladesh 12 c 3-6-15 Hikarigaoka, Munakata, Fukuoka 811-3403, Japan 13 d Graduate School of Human and Environmental Studies, Kyoto University, 14 Sakyo-ku, Kyoto 606-8501, Japan 15 16 *corresponding author 17 Phone: +81-82-424-7482 18 Fax: +81-82-424-0739 19 Email: [email protected] 20 21 1 1 Abstract 2 To elucidate the species composition, genetic divergence, evolutionary 3 relationships and divergence time of Hoplobatrachus and Euphlyctis frogs (subfamily 4 Dicroglossinae, family Ranidae) in Bangladesh and other Asian countries, we 5 analyzed the mitochondrial Cyt b, 12S and 16S rRNA genes of 252 specimens. Our 6 phylogenetic analyses showed 13 major clades corresponding to several cryptic 7 species as well as to nominal species in the two genera. The results suggested 8 monophyly of Asian Hoplobatrachus species, but the position of African H. 9 occipitalis was not clarified. Nucleotide divergence and phylogenetic data suggested 10 the presence of allopatric cryptic species allied to E. hexadactylus in Sundarban, 11 Bangladesh and several parapatric cryptic species in the Western Ghats, India. The 12 presence of at least two allopatric cryptic species among diverged E. cyanophlyctis in 13 Bangladesh, India and Sri Lanka was also suggested. In some cases, our estimated 14 divergence times matched the paleogeological events of South and Southeast Asian 15 regions that may have led to the divergence of Hoplobatrachus and Euphlyctis taxa. 16 Especially, Land formation at Bangladesh (15-10 Ma) may have allowed the spread of 17 these frog taxa to Southeast Asian areas, and the aridification of central India (5.1-1.6 18 Ma) might have affected the gene flow of widely distributed species. The present 19 study revealed prior underestimation of the richness of the amphibian fauna in this 20 region, indicating the possible occurrence of many cryptic species among these groups. 2 1 Key words: Genetic divergence; Molecular phylogeny; Mitochondrial genes; 2 Divergence time; Amphibia; Hoplobatrachus; Euphlyctis; Cryptic species; 3 Bangladesh 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 3 1 1. Introduction 2 Bangladesh, located in the tropical climatic zone, features one of the world’s 3 largest deltas (Ganges–Brahmaputra river delta) formed by Miocene sedimentation 4 and subsidence during continent-continent collision (Uddin and Lundberg, 2004) and 5 is endowed with a rich diversity of unique flora and fauna. Biogeographically, this 6 country is part of the Oriental region, nestled between the Indo-Himalayan and Indo- 7 Chinese subregions of the Orient (Nishat et al., 2002). Although the amphibian fauna 8 of the Western Ghats, India includes a large number of endemic taxa (Inger and Dutta, 9 1986), the available information on Bangladesh amphibian fauna lists only 22 frog 10 species (Islam et al., 2000). A recent herpetofaunal survey reported the occurrence of 11 some interesting species in Bangladesh for the first time (Reza et al., 2007), but the 12 genetic divergence and evolutionary aspects of the herpetofauna of Bangladesh have 13 basically been neglected. 14 Among the amphibian fauna reported from Bangladesh, Hoplobatrachus and 15 Euphlyctis frogs were the most common species, and during the 1980s Bangladesh 16 was a major world supplier of frogs. The Bangladesh Government eventually banned 17 the exporting of frogs in order to maintain the country’s natural resources and 18 ecological balance. As for the genus Hoplobatrachus, H. tigerinus (Indian bullfrog) is 19 one of the most widely distributed species in Bangladesh, whereas the distribution of 20 H. crassus (Jerdon’s bullfrog) is not clear due to insufficient data (Islam et al., 2000). 21 These two species are also distributed in other Asian countries such as India, Nepal, 4 1 Bhutan, and Sri Lanka (Frost, 2007). Two more species belonging to the genus 2 Hoplobatrachus are distributed in other countries: H. chinensis in Myanmar, China, 3 Thailand, and Malaysia, and H. occipitalis in several African countries (Frost, 2007). 4 As for the genus Euphlyctis, E. cyanophlyctis (Indian skipper frog) and E. 5 hexadactylus (Indian green frog) are known from Bangladesh (Islam et al., 2000). The 6 type localities of these two species are not clear, but Frost (2007) and Bauer (1998) 7 suggested that they might be in Tranquebar and Pondichéry located in Southeast 8 India near Sri Lanka. They also show wide distribution in other Asian countries: E. 9 cyanophlyctis in India, Pakistan, Afghanistan, Nepal, Sri Lanka, Myanmar, and 10 Vietnam, and E. hexadactylus in India, Pakistan, and Sri Lanka (Frost, 2007). Among 11 them, E. cyanophlyctis from the northwestern highlands of Pakistan was recognized as 12 a subspecies, E. cyanophlyctis microspinulata (Khan, 1997). Two more species 13 belonging to the genus Euphlyctis are distributed in other Asian countries: E. ghoshi, 14 known only from its type locality (Manipur, India), and E. ehrenbergii, inhabiting the 15 southwestern Arabian Peninsula (Saudi Arabia and Yemen) (Frost, 2007). 16 It is well known that the genus Hoplobatrachus is the sister taxon to the genus 17 Euphlyctis (Kosuch et al., 2001; Grosjean et al., 2004; Kurabayashi et al., 2005; Frost 18 et al., 2006). The species of these two genera were formerly regarded as members of 19 the genus Rana. However, Dubois (1987, 1992) suggested that the genus Rana was a 20 phylogenetically heterogeneous group, and transferred many species from Rana to 21 other genera including Hoplobatrachus and Euphlyctis. Although several studies have 5 1 been performed for phylogenetic analyses of higher taxa including these genera 2 (Bossuyt et al., 2006; Kosuch et al., 2001; Roelants et al., 2004; Vences et al., 2003), 3 there has been no investigation regarding detailed species composition, genetic 4 relationships and phylogeographic patterns among Hoplobatrachus and Euphlyctis 5 groups in Bangladesh and neighboring countries. 6 The increasing utilization of molecular data has led to the reorganization of 7 amphibian taxonomy (Biju and Bossuyt, 2003; Borkin et al., 2004; Bossuyt et al., 8 2006; De la Riva et al., 2000; Frost et al., 2006 Meegaskumbura et al., 2002) and the 9 discovery of many cryptic species (Bickford et al., 2006; Fouquet et al., 2007a, b; 10 Köhler et al., 2005; Stuart et al., 2006). Recent analyses of molecular and allozyme 11 data on samples from Asian countries suggested the underestimation of diversity of 12 amphibian fauna in this region as well as among these groups (Kurabayashi et al., 13 2005; Djong et al., 2007a, b; Kuramoto et al., 2007; Sumida et al., 2007; Islam et al., 14 2008). Inger (1999) suggested that additional samplings in South Asia would 15 undoubtedly increase the number of species known from each area and illuminate 16 detailed information on the distribution of species. 17 In order to elucidate the genetic diversity and phylogenetic relationships among 18 Hoplobatrachus and Euphlyctis groups from Bangladesh and neighboring countries, 19 we performed molecular phylogenetic analyses using mitochondrial Cyt b and 12S 20 and 16S rRNA gene data from 252 frog specimens. Based on the results, we showed 21 the possible existence of several cryptic species in these frog groups. We also 6 1 estimated the divergence times among these taxa to determine the paleogeological 2 events that had caused these divergences. 3 4 2. Materials and Methods 5 2.1. Specimens 6 A total of 252 individuals consisting of four species of the genus 7 Hoplobatrachus (H. tigerinus, H. crassus, H. chinensis, and H. occipitalis) and two 8 species of the genus Euphlyctis (E. cyanophlyctis and E. hexadactylus) were used in 9 the present study (Table 1, Fig. 1). Among them, 201 individuals were collected from 10 17 localities in Bangladesh, 46 individuals from 20 localities in India, Nepal, Sri 11 Lanka, Thailand, Laos, and Vietnam, and three individuals of H. occipitalis were 12 commercially obtained from Tanzania. Species identification was based on Dubois 13 (1992) and Frost (2007) classifications. Details of specimens are shown in electric 14 supplement 1. 15 16 2.2. DNA extraction 17 Total genomic DNA for PCR was extracted from the clipped toes of each 18 specimen using a DNA extraction kit (DNeasy Tissue Kit, QIAGEN) according to the 19 manufacturer’s instructions. The extracted DNA solutions were used to amplify partial 20 fragments of Cyt b and 12S and 16S rRNA genes by polymerase chain reaction (PCR). 21 7 1 2.3. PCR and sequencing 2 PCR amplification was performed on partial sequences of Cyt b (564 bp), 12S 3 rRNA (689 bp), and 16S rRNA (517 bp) genes. These segments corresponded to the 4 sites 16785–17348, 4474–5163, and 6251–6765, respectively, in the Fejervarya 5 limnocharis complete mtDNA sequence (Accession No. AY158705, Liu et al., 2005). 6 The following sets of primers were used for PCR amplification: Cytb Fow-1-1 (Sano 7 et al., 2005) and Cytb Rev-1 (Kurabayashi, unpublished) for Cyt b gene, FS01 and 8 RFR60 for 12S rRNA gene (Sumida et al., 1998), and F51 and R51 for 16S rRNA 9 gene (Sumida et al., 2002). The sequences of the primers are available from electric TM 10 supplement 2. PCR mixtures were prepared with the TaKaRa Ex Taq Kit (TaKaRa 11 Bio Inc.) as recommended by the manufacturer’s protocol.

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